RESUMEN
Advances in structural biology techniques over the last decades have made it increasingly possible to determine the structures of multi-protein complexes. Generation of sufficient recombinant material for such studies remains a bottleneck and often requires screening a variety of purification strategies and different subunit compositions to reproducibly isolate homogeneous complexes. Parallel advances in molecular biology now make it possible to easily generate panels of constructs with different affinity tags and different multi-protein components. Here, we describe two protocols based on Golden Gate cloning, which facilitate the generation of multi-protein complexes for protein production via the Baculovirus Expression Vector System. This robust method makes it possible to efficiently generate a panel of multi-gene expression constructs containing up to 15 open reading frames.
Asunto(s)
Vectores Genéticos , Proteínas , Baculoviridae/genética , Baculoviridae/metabolismo , Clonación Molecular , Vectores Genéticos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Microsporidios/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas Fúngicas , Insectos , Microsporidios/genética , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science.
Asunto(s)
Algoritmos , Estabilidad Proteica , Animales , Escherichia coli/metabolismo , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Proteínas/química , Proteínas/metabolismo , Solubilidad , Temperatura , Pez CebraRESUMEN
BACKGROUND: Recombinant protein production and purification of large protein complexes in eukaryotes requires efficient methods to generate multi-gene expression constructs, where each individual gene is under the control of its own promoter and terminator. Current methods are based either on serial rounds of combination of several vectors containing loxP sites via the Cre-lox technology, or on multiple rounds of gene combination via PCR or other methods. These methods are multi-step, have lower efficiencies than single gene cloning, and may require laborious processes to verify that all genes of interest are present in the final product. Here, we describe a rapid and simple Golden Gate-based system for the generation of multi-gene expression constructs compatible with baculovirus expression vector systems (BEVS) using either Tn7 transposition or KO1629-based homologous recombination, which we refer to as "GoldenBac". RESULTS: This method is based on the construction of a series of vectors containing a promoter-gene of interest-terminator cassette flanked by cleavage sites of the BsaI type IIS restriction enzyme. This series of vectors can be cut by BsaI to excise cassettes with unique overhangs. In the same reaction, the cassettes are then ligated in the correct sequence in a final destination vector to generate multi-gene expression constructs containing 2-15 genes. Individual expression constructs can therefore be combined into a single vector in a single reaction, with over 90% efficiency when combining up to 14 expression cassettes. We demonstrate successful construction and expression of three different co-expression systems, the proteosomal lid complex, the anaphase promoting complex/cyclosome (APC/C), and a series of constructs used to test the effect of chaperone co-expression on the solubility of the HOIP protein. CONCLUSIONS: This robust, single-step cloning system provides an easy-to-use method for generation of multi-gene expression constructs for both transposition and homologous recombination-based baculovirus systems, making this technology available across all laboratories using baculovirus expression systems. This highly efficient and simple method allows for rapid incorporation of multi-gene expression cloning into the standardized service portfolio of protein production facilities and can also easily be adopted by any laboratory for routine generation of multi-gene baculovirus constructs.
Asunto(s)
Baculoviridae/genética , Clonación Molecular/métodos , Expresión Génica , Proteínas Recombinantes/genética , Animales , Enzimas de Restricción del ADN/genética , Escherichia coli/genética , Técnicas de Inactivación de Genes , Vectores Genéticos , Proteínas de Choque Térmico , Recombinación Homóloga , Reacción en Cadena de la Polimerasa , Células Sf9RESUMEN
Plants use surface receptors to perceive information about many aspects of their local environment. These receptors physically interact to form both steady state and signalling competent complexes. The signalling events downstream of receptor activation impact both plant developmental and immune responses. Here, we present a comprehensive study of the physical interactions between the extracellular domains of leucine-rich repeat receptor kinases (LRR-RKs) in Arabidopsis. Using a sensitized assay, we tested reciprocal interactions among 200 of the 225 Arabidopsis LRR-RKs for a total search space of 40,000 interactions. Applying a stringent statistical cut-off and requiring that interactions performed well in both bait-prey and prey-bait orientations resulted in a high-confidence set of 567 bidirectional interactions. Additionally, we identified a total of 2,586 unidirectional interactions, which passed our stringent statistical cut-off in only one orientation. These datasets will guide further investigation into the regulatory roles of LRR-RKs in plant developmental and immune signalling decisions.
Asunto(s)
Proteínas de Arabidopsis , Mapeo de Interacción de Proteínas , Proteínas Quinasas/química , Proteínas , Proteínas de Arabidopsis/química , Proteínas Repetidas Ricas en Leucina , Dominios Proteicos , Mapeo de Interacción de Proteínas/métodos , Proteínas Quinasas/fisiologíaRESUMEN
Resolving functions of closely linked genes is challenging or nearly impossible with classical genetic tools. Four members of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) family are clustered on Arabidopsis chromosome five. To resolve the potentially redundant functions of this subclass of CrRLK1Ls named MEDOS1 to 4 (MDS1 to 4), we generated a single CRISPR/Cas9 transformation vector using a Golden Gate based cloning system to target all four genes simultaneously. We introduce single mutations within and deletions between MDS genes as well as knock-outs of the whole 11 kb gene cluster. The large MDS cluster deletion was inherited in up to 25% of plants lacking the CRISPR/Cas9 construct in the T2 generation. In contrast to described phenotypes of already characterized CrRLK1L mutants, quadruple mds knock-outs were fully fertile, developed normal root hairs and trichomes and responded to pharmacological inhibition of cellulose biosynthesis similar to wildtype. Recently, we demonstrated the role of four CrRLK1L in growth adaptation to metal ion stress. Here we show the involvement of MDS genes in response to Ni2+ during hypocotyl elongation and to Cd2+ and Zn2+ during root growth. Our finding supports the model of an organ specific network of positively and negatively acting CrRLK1Ls.
Asunto(s)
Arabidopsis/genética , Catharanthus/genética , Ingeniería Genética/métodos , Proteínas de Plantas/genética , Proteínas de Arabidopsis/genética , Sistemas CRISPR-Cas , Cationes , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genoma de Planta , Metales/farmacología , Familia de Multigenes , Mutagénesis , Mutación , Fenotipo , Plantas Modificadas Genéticamente/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
In this Letter, an incorrect version of the Supplementary Information file was inadvertently used, which contained several errors. The details of references 59-65 were missing from the end of the Supplementary Discussion section on page 4. In addition, the section 'Text 3. Y2H on ICD interactions' incorrectly referred to 'Extended Data Fig. 4d' instead of 'Extended Data Fig. 3d' on page 3. Finally, the section 'Text 4. Interaction network analysis' incorrectly referred to 'Fig. 1b and Extended Data Fig. 6' instead of 'Fig. 2b and Extended Data Fig. 7' on page 3. These errors have all been corrected in the Supplementary Information.
RESUMEN
Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204â¯kDa; human ABL1 wildtype, 126â¯kDa; human FMRP, 68â¯kDa; viral vNS1-H1, 76â¯kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.
Asunto(s)
Baculoviridae/genética , Vectores Genéticos/genética , Proteínas Recombinantes/metabolismo , Animales , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Humanos , Ratones , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Recombinantes/genética , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Células Sf9RESUMEN
The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, we investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay, and produced an LRR-based cell surface interaction network (CSILRR) that consists of 567 interactions. To demonstrate the power of CSILRR for detecting biologically relevant interactions, we predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, we show that CSILRR operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Leucina/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Arabidopsis/citología , Arabidopsis/inmunología , Arabidopsis/microbiología , Unión Proteica , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Reproducibilidad de los Resultados , Transducción de SeñalRESUMEN
Telomeres form specialized chromatin that protects natural chromosome termini from being recognized as DNA double-strand breaks. Plants possess unusual blunt-ended telomeres that are unable to form t-loops or complex with single-strand DNA binding proteins, raising the question of the mechanism behind their protection. We have previously suggested that blunt-ended telomeres in Arabidopsis thaliana are protected by Ku, a DNA repair factor with a high affinity for DNA ends. In nonhomologous end joining, Ku loads onto broken DNA via a channel consisting of positively charged amino acids. Here, we demonstrate that while association of Ku with plant telomeres also depends on this channel, Ku's requirements for DNA binding differ between DNA repair and telomere protection. We show that a Ku complex proficient in DNA loading but impaired in translocation along DNA is able to protect blunt-ended telomeres but is deficient in DNA repair. This suggests that Ku physically sequesters blunt-ended telomeres within its DNA binding channel, shielding them from other DNA repair machineries.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , ADN de Plantas/genética , Autoantígeno Ku/metabolismo , Telómero/genética , Reparación del ADN/genética , Autoantígeno Ku/genéticaRESUMEN
We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.
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Proteínas Fluorescentes Verdes/biosíntesis , Complejos Multiproteicos/biosíntesis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Virales/biosíntesis , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Técnicas de Cultivo de Célula , Transferencia Resonante de Energía de Fluorescencia/métodos , Código Genético , Vectores Genéticos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.
Asunto(s)
Proteínas Recombinantes/aislamiento & purificación , Automatización de Laboratorios , Cromatografía en Gel/métodos , Clonación Molecular , Clonación de Organismos , Biología Computacional , Escherichia coli/genética , Escherichia coli/metabolismo , Filaminas/genética , Filaminas/aislamiento & purificación , Proteínas Recombinantes/biosíntesisRESUMEN
Oscillating cyclin-dependent kinase 1 (Cdk1) activity is the major regulator of cell-cycle progression, whereas the Aurora B kinase, as part of the chromosome passenger complex (CPC), controls critical aspects of mitosis such as chromosome condensation and biorientation on the spindle. How these kinases mechanistically coordinate their important functions is only partially understood. Here, using budding yeast, we identify a regulatory mechanism by which the Cdk1 kinase Cdc28 directly controls the Aurora kinase Ipl1. We show that Cdk1 phosphorylates Ipl1 on two serine residues in the N-terminal domain, thereby suppressing its association with the microtubule plus-end tracking protein Bim1 until the onset of anaphase. Failure to phosphorylate Ipl1 leads to its premature targeting to the metaphase spindle and results in constitutive Bim1 phosphorylation, which is normally restricted to anaphase. Cells expressing an Ipl1-Sli15 complex that cannot be phosphorylated by Cdk1 display a severe growth defect. Our work shows that Ipl1/Aurora is not only the catalytic subunit of the CPC but also an important regulatory target that allows Cdk1 to coordinate chromosome biorientation with spindle morphogenesis.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Aurora Quinasas , FosforilaciónRESUMEN
Deg1 is a chloroplastic protease involved in maintaining the photosynthetic machinery. Structural and biochemical analyses reveal that the inactive Deg1 monomer is transformed into the proteolytically active hexamer at acidic pH. The change in pH is sensed by His244, which upon protonation, repositions a specific helix to trigger oligomerization. This system ensures selective activation of Deg1 during daylight, when acidification of the thylakoid lumen occurs and photosynthetic proteins are damaged.
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Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Luz , Complejo de Proteína del Fotosistema II/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Arabidopsis/efectos de la radiación , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The peptide transporter (PTR) family represents a group of proton-coupled secondary transporters responsible for bulk uptake of amino acids in the form of di- and tripeptides, an essential process employed across species ranging from bacteria to humans. To identify amino acids critical for peptide transport in a prokaryotic PTR member, we have screened a library of mutants of the Escherichia coli peptide transporter YdgR using a high-throughput substrate uptake assay. We have identified 35 single point mutations that result in a full or partial loss of transport activity. Additional analysis, including homology modeling based on the crystal structure of the Shewanella oneidensis peptide transporter PepT(so), identifies Glu(56) and Arg(305) as potential periplasmic gating residues. In addition to providing new insights into transport by members of the PTR family, these mutants provide valuable tools for further study of the mechanism of peptide transport.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas Periplasmáticas , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Shewanella/química , Shewanella/genética , Shewanella/metabolismo , Homología Estructural de ProteínaRESUMEN
The serotonin transporter (SERT) is a member of the SLC6 family of solute carriers. SERT plays a crucial role in synaptic neurotransmission by retrieving released serotonin. The intracellular carboxyl terminus of various neurotransmitter transporters has been shown to be important for the correct delivery of SLC6 family members to the cell surface. Here we studied the importance of the C terminus in trafficking and folding of human SERT. Serial truncations followed by mutagenesis identified sequence spots (PG(601,602), RII(607-609)) within the C terminus relevant for export of SERT from the endoplasmic reticulum (ER). RI(607,608) is homologous to the RL-motif that in other SLC6 family members provides a docking site for the COPII component Sec24D. The primary defect resulting from mutation at PG(601,602) and RI(607,608) was impaired folding, because mutated transporters failed to bind the inhibitor [(3)H]imipramine. In contrast, when retained in the ER (e.g. by dominant negative Sar1) the wild type transporter bound [(3)H]imipramine with an affinity comparable to that of the surface-expressed transporter. SERT-RI(607,608)AA and SERT-RII(607-609)AAA were partially rescued by treatment of cells with the nonspecific chemical chaperone DMSO or the specific pharmacochaperone ibogaine (which binds to the inward facing conformation of SERT) but not by other classes of ligands (inhibitors, substrates, amphetamines). These observations (i) demonstrate an hitherto unappreciated role of the C terminus in the folding of SERT, (ii) indicates that the folding trajectory proceeds via an inward facing intermediate, and (iii) suggest a model where the RI-motif plays a crucial role in preventing premature Sec24-recruitment and export of incorrectly folded transporters.
Asunto(s)
Mutación , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Imipramina/química , Ligandos , Proteínas de la Membrana/química , Microscopía Fluorescente/métodos , Chaperonas Moleculares/química , Neurotransmisores/química , Pliegue de Proteína , Estructura Terciaria de Proteína , Serotonina/químicaRESUMEN
Membrane proteins comprise up to one-third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. Membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. We are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. Here, we present the comparative expression data for 37 target proteins, all of them secondary transporters, from the mesophilic organism Salmonella typhimurium and the two hyperthermophilic organisms Aquifex aeolicus and Pyrococcus furiosus in three different Escherichia coli expression vectors. In addition, we study the use of Lactococcus lactis as a host for integral membrane protein expression. Overall, 78% of the targets were successfully produced under at least one set of conditions. Analysis of these results allows us to assess the role of different variables in increasing "expression space" coverage for our set of targets. This analysis implies that to maximize the number of nonhomologous targets that are expressed, orthologous targets should be chosen and tested in two vectors with different types of promoters, using C-terminal tags. In addition, E. coli is shown to be a robust host for the expression of prokaryotic transporters, and is superior to L. lactis. These results therefore suggest appropriate strategies for high-throughput heterologous overproduction of membrane proteins.